Project 2: Translocations of p66Shc: kinetics, function, apoptosis The overall goal of this project is to understand the mechanism that drives the organellar translocation of p66Shc, how p66Shc influences mitochondrial parameters once it arrives there, and how these trigger apoptosis and then to design inhibitors of this translocation process as de facto p66Shc inhibitors/Thus, Specific Aims for this project are to: (1) Identify the link between reactive oxygen species exposure and She recruitment; we will identify (i) the upstream kinases leading to the phosphorylation of Ser36 and (ii) the cellular systems that recognize and activate the phosphorylated protein. We expect that peptidyl-prolyl-isomerase recognizes the phosphorylation of p66Shc and therefore is critical for the apoptotic effect. (2) Measure stress-dependent translocation of p66shc to mitochondria. We will identify the mechanism, timing, and regulation of the translocation of p66shcto the mitochondrial inter-membrane space; we will evaluate the localization of p66Shc to the ER-mitochondrial contact sites and the interaction of p66Shc with other molecules we have previously demonstrated to be involved in ER-to-mitochondrial signaling. (3) Investigate the consequences of p66Shc activation on mitochondria. We will clarify the consequences of p66shc activation on mitochondria, including (i) mitochondrial calcium homeostasis, (ii) mitochondrial membrane potential, (iii) 3D mitochondrial structure, and (iv)ATP production. (4) Examine the mechanism of apoptosis following p66Shc activation. We will test the participation of two basic mitochondrial mechanisms of apoptosis induction, measure mitochondrial depolarization as a hallmark of mitochondrial permeability transition (MPT), and then evaluate the interaction of p66Shc pathways with Bcl-2 pathways by coexpressing isoforms of each. (5) Develop novel tools to identify drugs acting on the She signaling pathway; we will develop two cell-based assays, suitable for use in high-throughput screenings, to address the potential for She-like therapeutics. Relevance to public health: Because the p66Shc pathway appears to be related to the IGF-lnsR pathway of lifespan extension that extends lifespan from flies to mice, and because p66Shc appears to control adipogenesis and may be related to metabolic syndrome, it is likely that our experiments and search for p66Shc recruitment inhibitors will be relevant to public health.